The involvement of free radical-induced oxidative damage to mitochondrial (mt) proteins and DNA has been suggested as a cause of the "vicious cycle" that results in mt dysfunction in age-related pathologies and neurodegenerative diseases. However, it has recently been shown that the levels of oxidative damage in mt RNA may be greater than in mt DNA, and that the accumulation of defective transcripts may be a major cause of mt dysfunction. To maintain the quality of transcripts mitochondria employ an extensive system of posttranscriptional RNA surveillance and must balance the rates of RNA synthesis and degradation in order to prevent swamping of this system. However, the molecular mechanisms that might adjust the level of transcription under various conditions are not known. One approach to this question is to identify proteins that are associated with the mt transcription complex under various conditions, and to examine their effects on mt RNAP activity in a purified in vitro system. In preliminary experiments, proteins that are associated with the yeast mt RNAP were successfully identified by affinity pulldown of TAP-tagged mt RNAP from isolated organelles. One of the most abundant proteins identified was Mss116p, which had previously been implicated in coordinating rates of RNA degradation and synthesis by genetic studies. Strikingly, we found that Mss116p enhances the stability of transcription complexes in vitro, and reduces the concentration of nucleoside triphosphate that is required to extend RNA from a paused complex. These effects are similar to those of elongation factors for multisubunit RNAPs and suggest that Mss116p might act to decrease the abundance of aberrant transcripts by allowing mt RNAP to complete RNA synthesis more efficiently, especially when the abundance of NTP substrates is limited, as would occur under oxidative stress conditions or oxygen/energy deprivation. Our preliminary results in the yeast system demonstrate the utility of this approach in identifying protein factors that may modulate mt transcription in mammalian. In the proposed studies we intend to apply this approach to identify protein factors that interact with the mt RNAP in neural tissue under normal and stress conditions using cultured rat primary cortical astrocytes (RPCA) as a model system, and to define their genome-wide localization and molecular mechanism of action by in vitro methods. Characterization of mt transcriptional plasticity in neural tissue may allow the delivery of new targets of potential therapies to decrease the rate of age-associated neural cell death. PUBLIC HEALTH RELEVANCE: These studies will provide insight as to how mitochondria respond to oxidative damage and to aging, and will allow identification of cis- and trans-acting factors that regulate the activity of mt genome and may serve as targets of longevity-improving interventions and therapies.